Effect of Aldosterone on MMP-9 Activity in Monocyte/Macrophages

Background. Atherosclerosis is a chronic, multifactorial pathology caused by long exposure to cardiovascular risk factors. Risk factors provoke oxidative stress and endothelial dysfunction, leading to expression of adhesion molecules, diapedesis of monocytes and transformation into macrophages. Macrophages internalize oxidized-LDL, become foam cells and release proinflammatory cytochines that increase atherosclerosis and metalloproteases that destabilize the plaque. High plasma aldosterone level (PAC) was shown to predict short and long-term mortality and life-threatening arrhythmias in patients with myocardial infarction (AMI) and a prognostic role was attributed to PAC also in stable coronary artery disease (CAD) patients. Moreover, mineralocorticoid receptor antagonism (MRA) was demonstrated to reduce cardiovascular (CV) mortality in patients with heart failure (HF) or left ventricular dysfunction post-acute AMI. Recent clinical trials demonstrated a direct aldosterone pro-atherogenic or plaque-destabilizing effect, supported by the recent identification of pathways linking aldosterone to oxidative stress and metalloproteinase-9 (MMP-9) activation, which we postulate might occur in monocytic/macrophagic cells, recognized effectors of plaque instability and ensuing CV events. Aim. The primary aim of our study was to investigate the effects of aldosterone and mineralocorticoid receptor antagonist on the MMP-9 production by monocytes/macrophages Material and Methods. We employ a THP-1 monocytic cell-line and human monocytes from blood of healthy donors; to obtain macrophages, we used phorbol myristate acetate (PMA) for the cell line and GM-CSF for human cells. We first confirmed the cell transition by FACS and gene expression of PPARγ gene. Cells were investigated for the presence of the Mineralocorticoid Receptor (MR) at Western Blot and RT-PCR and to elucidate the effects of aldosterone on these cells we analyzed FKBP51 gene expression. The effect on MMP-9 expression and activity, was assessed by RT-PCT and zimography, respectively. Results. We demonstrated that macrophages, both THP-1 and human, express an higher amount of PPARγ mRNA, confirming the successful phenotypic transition. Macrophages express also an higher amount of MR protein and mRNA respect to monocytes, thus explaining the non-responsiveness of monocytes to aldosterone in MMP-9 increase. On the contrary, THP-1 macrophages (differentiated after 72 hours in PMA 100nM), showed in the dose and time-dependent curve, a significant MMP-9 activity in response to aldosterone in particular at lower concentrations (10-8 and 10-9M) between 16 and 48 hours. Responsiveness of macrophages to aldosterone was confirmed by FKBP51 gene expression. We repeated the dose and time-response curve in human cells: MMP-9 gene expression was significantly higher after 2 hours of aldosterone at 10-7M and 10-8M (2,03 ± 0,26 and 2,05 ± 0,26 fold change vs. control, respectively; p < 0.001) and at 6 hours only aldosterone at 10-7M, while MMP-9 activity increases only at 24 hours in presence of aldosterone 10-8 M aldosterone 10-9 M (1,23 ± 0,07 and 1,36 ± 0,08 respectively). In presence of canrenone as MR inhibitor, RT-PCR shows a clear trend in reduction both at 2 and 6 hours in presence of canrenone respect to aldosterone-induced MMP-9 gene expression, even if not significant (p = 0.08 at 6 hours)